Multimedia resources such as video and animations are increasingly used to enhance student engagement and understanding, particularly when teaching cognitively complex concepts. However, the creation of animation is time-consuming and hence, expensive compared to the creation of graphics. Recognizing this and the challenges students face in learning immunology, we describe here a process of a multi-disciplinary collaboration that produced a series of 3-minute animated infographics videos for tertiary-level immunology teaching within an Australian university. We evaluate the benefit of these and their merit as supplemental curriculum resources to enhance learning.

RNA interference (RNAi) is a mechanism conserved in eukaryotes, including fungi, that represses gene expression by means of small noncoding RNAs (sRNAs) of about 20 to 30 nucleotides. Its discovery is one of the most important scientific breakthroughs of the past 20 years, and it has revolutionized our perception of the functioning of the cell. Initially described and characterized in Neurospora crassa, the RNAi is widespread in fungi, suggesting that it plays important functions in the fungal kingdom. Several RNAi-related mechanisms for maintenance of genome integrity, particularly protection against exogenous nucleic acids such as mobile elements, have been described in several fungi, suggesting that this is the main function of RNAi in the fungal kingdom. However, an increasing number of fungal sRNAs with regulatory functions generated by specific RNAi pathways have been identified. Several mechanistic aspects of the biogenesis of these sRNAs are known, but their function in fungal development and physiology is scarce, except for remarkable examples such as Mucor circinelloides, in which specific sRNAs clearly regulate responses to environmental and endogenous signals. Despite the retention of RNAi in most species, some fungal groups and species lack an active RNAi mechanism, suggesting that its loss may provide some selective advantage. This article summarizes the current understanding of RNAi functions in the fungal kingdom.

A key determinant for the survival of organisms is their capacity to recognize and respond efficiently to foreign antigens. This is largely accomplished by the orchestrated activity of the innate and adaptive branches of the immune system. Antibodies are specifically generated in response to foreign antigens, facilitating thereby the specific recognition of antigens of almost infinite diversity. Receptors specific for the Fc domain of antibodies, Fc receptors, are expressed on the surface of the various myeloid leukocyte populations and mediate the binding and recognition of antibodies by innate leukocytes. By directly linking the innate and the adaptive components of immunity, Fc receptors play a central role in host defense and the maintenance of tissue homeostasis through the induction of diverse proinflammatory, anti-inflammatory, and immunomodulatory processes that are initiated upon engagement by the Fc domain. In this chapter, we discuss the mechanisms that regulate Fc domain binding to the various types of Fc receptors and provide an overview of the astonishing diversity of effector functions that are mediated through Fc-FcR interactions on myeloid cells. Lastly, we discuss the impact of FcR-mediated interactions in the context of IgG-mediated inflammation, autoimmunity, susceptibility to infection, and responsiveness to antibody-based therapeutics.

Citation: Poirier V, Av-Gay Y. 2015. Intracellular growth of bacterial pathogens: the role of secreted effector proteins in the control of phagocytosed microorganisms. 3(6): doi:10.1128/microbiolspec.VMBF-0003-2014

The ability of intracellular pathogens to subvert the host response, to facilitate invasion and subsequent infection, is the hallmark of microbial pathogenesis. Bacterial pathogens produce and secrete a variety of effector proteins, which are the primary means by which they exert control over the host cell. Secreted effectors work independently, yet in concert with each other, to facilitate microbial invasion, replication, and intracellular survival in host cells. In this review we focus on defined host cell processes targeted by bacterial pathogens. These include phagosome maturation and its subprocesses: phagosome-endosome and phagosome-lysosome fusion events, as well as phagosomal acidification, cytoskeleton remodeling, and lysis of the phagosomal membrane. We further describe the mode of action for selected effectors from six pathogens: the Gram-negative Legionella, Salmonella, Shigella, and Yersinia, the Gram-positive Listeria, and the acid-fast actinomycete Mycobacterium.

For a generation of microbiologists who study pathogenesis in the context of the human microbiome, understanding the diversity of bacterial metabolism is essential. In this chapter, I briefly describe how and why I became, and remain, interested in metabolism. I then will describe and compare some of the strategies used by bacteria to consume sugars as one example of metabolic diversity. I will end with a plea to embrace metabolism in the endeavor to understand pathogenesis.

Hairpin telomere resolvases (also known as protelomerases) have emerged as a unique solution to the end replication problem (1, 2). These enzymes promote the formation of covalently closed hairpin ends on linear DNA molecules in some phage (3, 4, 5), bacterial plasmids and bacterial chromosomes (6, 7, 8, 9). Telomere resolvases are mechanistically related to tyrosine recombinases and type IB topoisomerases and are also believed to play a role in the genome plasticity that characterizes Borrelia species. Fig. 1 shows the reaction pathway for replication of linear DNA molecules with covalently closed hairpin telomeres. Duplication of the DNA molecule results in replicated telomeres (rTel, also referred to as dimer junctions) that are recognized and processed in a DNA breakage and reunion reaction promoted by a hairpin telomere resolvase. The reaction products are covalently closed hairpin telomeres at both ends of linear monomeric DNA molecules. At this writing telomere resolvases have been purified from three phage and seven bacterial species: E. coli phage N15 (3), Klebsiella oxytoca phage ɸKO2, Yersinia enterocolitica phage PY54 (5), Agrobacterium tumefaciens (8), the Lyme spirochete Borrelia burgdorferi (6), the relapsing fever borreliae B. hermsii, B. parkeri, B. recurrentis, B. turicatae, and the avian spirochete B. anserina (7). The B. burgdorferi enzyme, ResT (Resolvase of Telomeres) has been the most extensively studied at the biochemical level (6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) and is the primary focus of this review, with properties of the other enzymes noted (3, 4, 5, 8, 24). Structural studies of the Klebsiella phage ɸKO2 (25) and the Agrobacterium (26) resolvases have been reported and have shed additional light on reaction mechanisms and on differences between the resolvases from different organisms.

Students often self-identify as visual learners and prefer to engage with a topic in an active, hands-on way. Indeed, much research has shown that students who actively engage with the material and are engrossed in the topics retain concepts better than students who are passive receivers of information. However, much of learning life science concepts is still driven by books and static pictures. One concept students have a hard time grasping is how a linear chain of amino acids folds to becomes a 3D protein structure. Adding three dimensional activities to the topic of protein structure and function should allow for a deeper understanding of the primary, secondary, tertiary, and quaternary structure of proteins and how proteins function in a cell. Here, I review protein folding activities and describe using Apps and 3D visualization to enhance student understanding of protein structure.

Covalently closed hairpin ends, also known as hairpin telomeres, provide an unusual solution to the end replication problem. The hairpin telomeres are generated from replication intermediates by a process known as telomere resolution. This is a DNA breakage and reunion reaction promoted by hairpin telomere resolvases (also referred to as protelomerases) found in a limited number of phage and bacteria. The reaction promoted by these enzymes is a chemically isoenergetic two-step transesterification without a requirement for divalent metal ions or high-energy cofactors and uses an active site and mechanism similar to that for type IB topoisomerases and tyrosine recombinases. The small number of unrelated telomere resolvases characterized to date all contain a central, catalytic core domain with the active site, but in addition carry variable C- and N-terminal domains with different functions. Similarities and differences in the structure and function of the telomere resolvases are discussed. Of particular interest are the properties of the Borrelia telomere resolvases, which have been studied most extensively at the biochemical level and appear to play a role in shaping the unusual segmented genomes in these organisms and, perhaps, to play a role in recombinational events.

The highly conserved Nus factors of bacteria were discovered as essential host proteins for the growth of temperate phage λ in Escherichia coli. Later, their essentiality and functions in transcription, translation, and, more recently, in DNA repair have been elucidated. Close involvement of these factors in various gene networks and circuits is also emerging from recent genomic studies. We have described a detailed overview of their biochemistry, structures, and various cellular functions, as well as their interactions with other macromolecules. Towards the end, we have envisaged different uncharted areas of studies with these factors, including their participation in pathogenicity.

Endospore formation follows a complex, highly regulated developmental pathway that occurs in a broad range of Firmicutes. Although Bacillus subtilis has served as a powerful model system to study the morphological, biochemical, and genetic determinants of sporulation, fundamental aspects of the program remain mysterious for other genera. For example, it is entirely unknown how most lineages within the Firmicutes regulate entry into sporulation. Additionally, little is known about how the sporulation pathway has evolved novel spore forms and reproductive schemes. Here, we describe endospore and internal offspring development in diverse Firmicutes and outline progress in characterizing these programs. Moreover, comparative genomics studies are identifying highly conserved sporulation genes, and predictions of sporulation potential in new isolates and uncultured bacteria can be made from these data. One surprising outcome of these comparative studies is that core regulatory and some structural aspects of the program appear to be universally conserved. This suggests that a robust and sophisticated developmental framework was already in place in the last common ancestor of all extant Firmicutes that produce internal offspring or endospores. The study of sporulation in model systems beyond B. subtilis will continue to provide key information on the flexibility of the program and provide insights into how changes in this developmental course may confer advantages to cells in diverse environments.

It is probably hard to find an educator who would hesitate to agree that abstract thinking and comfort in asking questions are pivotal to scientific inquiry and advancement of knowledge. Yet, most of the time the mechanics of fostering these skills is as challenging as photographing dense fog. As biologists we constantly reevaluate what we know, how we think about what we know, and how we communicate our knowledge about the living world. These short engaging exercises challenge students to appreciate the central role of abstract thinking and question asking in scientific inquiry. In the first exercise the classroom is presented with an optical illusion image and challenged to evaluate it using concrete and abstract thinking tools. In a follow up exercise, students are prompted to evaluate the process of making assumptions, asking questions and coming to conclusions using the example of a small popular culture article. Both exercises are used as primers to stimulate discussion emphasizing that abstract thinking and question asking are critical tools in the tool set of 21st century budding biologists.

Examination of the transcriptome and proteome enables the investigation of the underlying gene and protein expression, respectively, that results in cold adaptation and ultimately permits the successful colonization of cold environments by cold-adapted microorganisms. Genomics can be used to investigate cold adaptation at the level of whole genes by examining gene content, gene expression, protein expression, and other unique features, while at the molecular level, genomic analyses may identify trends in amino acid composition, codon usage, and nucleotide content that result from cold adaptation. This chapter discusses (i) use of ecological information to discern cold-adapted microorganisms, (ii) unique gene- and protein-expression adaptations for coping with cold environment stresses, (iii) sequence adaptations that facilitate protein function at low temperature, and (iv) a case study comparing cold-adapted and warm-adapted species of the genus Exiguobacterium. The genera Exiguobacterium and Psychrobacter represent gram-positive and gram-negative bacteria, respectively. Strains of these two genera were among the psychrophile genomes sequenced and used, along with other examples, to illustrate various aspects of cold adaptation. Five prominent eurypsychrophiles including the permafrost firmicute E. sibiricum 255-15 have been subjected to functional genomics experimentation at low temperature. Findings from studies with these organisms with reference to other psychrophilic and mesophilic microbes where appropriate, are presented in the chapter. The results suggested that E. sibiricum requires active transport of nutrients at lower temperature to increase substrate uptake.

Homologous recombination is the most complex of all recombination events that shape genomes and produce material for evolution. Homologous recombination events are exchanges between DNA molecules in the lengthy regions of shared identity, catalyzed by a group of dedicated enzymes. There is a variety of experimental systems in Escherichia coli and Salmonella to detect homologous recombination events of several different kinds. Genetic analysis of homologous recombination reveals three separate phases of this process: pre-synapsis (the early phase), synapsis (homologous strand exchange), and post-synapsis (the late phase). In E. coli, there are at least two independent pathway of the early phase and at least two independent pathways of the late phase. All this complexity is incongruent with the originally ascribed role of homologous recombination as accelerator of genome evolution: there is simply not enough duplication and repetition in enterobacterial genomes for homologous recombination to have a detectable evolutionary role and therefore not enough selection to maintain such a complexity. At the same time, the mechanisms of homologous recombination are uniquely suited for repair of complex DNA lesions called chromosomal lesions. In fact, the two major classes of chromosomal lesions are recognized and processed by the two individual pathways at the early phase of homologous recombination. It follows, therefore, that homologous recombination events are occasional reflections of the continual recombinational repair, made possible in cases of natural or artificial genome redundancy.

The largest class of small RNAs (sRNAs) regulate mRNA stability and translation by pairing with specific target mRNAs. The way in which the mRNA folds can profoundly affect all of its properties. Degradation by RNase E in vitro is known to be stimulated by a 5' monophosphate or 5' OH on the mRNA, even though the initial cleavage event may be far from the 5' end. Initiation of degradation at the 3' end of an mRNA without an internal endonucleolytic cut generally proceeds rapidly only in the absence of secondary structure, such as the stem loop from a factor-independent terminator. Most alterations in mRNA folding by regulatory molecules affect stability or translation, but co-transcriptional changes in folding can also affect mRNA fate via formation or failure to form RNA structures, thereby leading to transcription termination. More recently, it has become apparent that environmental sensing by the 5' end of an mRNA can occur directly, by binding of a small molecule ligand to the 5' UTR. The folding of these mRNA structures, called riboswitches, is modulated by the ligand, leading to transcription termination (OFF switches) or antitermination (ON switches), or translational regulation. Total deletion of CsrA is lethal in Escherichia coli under many growth conditions, apparently because of redirection of the cell to hyperaccumulation of glycogen. Antisense RNAs, known to regulate plasmid stability, have now been found in bacteria and shown to play major regulatory roles.

This chapter highlights some of the advances on both the molecular mechanisms of oxygen (O2) sensing and the biological responses to O2 limitation. In the first part of the chapter, the major regulators that control expression of anaerobic respiratory pathways are described, with focus on the well studied examples from Escherichia coli K-12. Many facultative bacteria have anaerobic lifestyles that depend on pathways not found in enteric bacteria. A section reviews the master regulators of these anaerobic lifestyles, describing how their activity responds to O2 deprivation, highlighting commonalities and differences to the responses described for enterobacteria, and placing them into a metabolic context of the systems they control. A recurring theme in the review is that multiple transcription factors collaborate in a given organism to control gene expression in response to changes in O2. In E. coli, decreased expression of the genes encoding aerobic respiratory functions under anaerobic growth conditions is largely mediated by the aerobic respiration control (Arc) A and ArcB two-component system. Regulation of anaerobic respiration in the γ-proteobacterium Shewanella oneidensis has attracted great interest because of the broad diversity of electron acceptors (>14) that these bacteria can respire, including metal oxides. The use of cofactors such as flavins, heme, and [Fe-S] clusters generally sense O2 directly, using chemistry reflecting their well-described roles in biological reactions.

Until the late 1940s, little was known about cryptococcal capsule composition or structure. Classically, the capsule is described as composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins, based on the original fractionation of shed polysaccharide material. Cell wall polymers are involved in capsule association with the cell, although they are not considered part of the capsule itself. Polysaccharide synthesis starts with the generation of precursor molecules, the most common being the activated sugars discovered by Leloir. A number of proteins involved in the synthesis of these precursors have been identified and studied in Cryptococcus neoformans. This work has been facilitated by the fact that many of these enzymes are highly conserved in terms of sequence, as would be expected from the participation of activated sugar precursors in multiple synthetic pathways across biology. In support of a lumenal location for capsule synthesis, a conditional mutant generated in both serotypes A and D that is defective in vesicle targeting to the plasma membrane accumulates post-Golgi vesicles containing GXM. The conclusion from this study is that GXM is made within the classical secretory pathway, consistent with the requirement for nucleotide sugar transporters to achieve normal capsule synthesis.

Signal transduction cascades are utilized by all organisms to convey signals perceived at the cell surface to effectors within the cell. These enzymatic signaling cascades are important in the pathogenesis of many infections, including cryptococcosis. This chapter summarizes the significance and functional interactions involved in the cell wall integrity, phospholipase, and calcineurin signaling pathways for the establishment of Cryptococcus neoformans virulence. The fungal Plc enzymes referred to in this review preferentially hydrolyze phosphatidylinositol (PI)-based substrates within the cryptococcal cell and affect multiple cellular functions, including the secretion of (phospholipase B ) Plb1. It was found that the Plb1 MW could be as high as 125 kDa due to extensive asparagine N-linked glycosylation, which is responsible for at least 30% of the MW of Plb1 and essential for its activity. It was recently demonstrated that PI-PLC1 (Plc1) regulates cryptococcal virulence, acting in part through interactions with the Pkc/Mpk1 cell wall integrity pathway. In contrast to Plcs from higher eukaryotes, Plcs from the parasite Trypanosoma brucei preferentially hydrolyze the glycosylphosphatidylinositol (GPI) anchor of variant surface glycoprotein or GPI biosynthetic intermediates, in addition to PI, but not the phosphorylated intermediates, despite their localization to the peripheral cytoplasmic face of intracellular vesicles. Metabolic labeling studies performed in S. cerevisiae implicated a Plc enzyme and a secondary-acting protease in hydrolysis of the GPI anchor of certain proteins in the plasma membrane, resulting in their subsequent localization in the cell wall. ScPlc1, the only Plc1 in S. cerevisiae, like CnPlc1, lacks a secretory signal leader peptide.

Viruses are the most abundant infectious agents on earth and the most primitive form of life, predating us by billions of years. Due to the fact that viruses utilize host cell machinery to replicate, the host has the daunting task of distinguishing virus infection signatures from self-molecules. This chapter provides an overview of the mechanisms used to sense virus infection (innate recognition), the cytokine system that evolved to rapidly induce antiviral states in neighboring cells (type I interferons), and the methods used to contain and destroy viruses (effector functions). Analysis of animals deficient in the RIG-I-like receptors (RLRs) revealed important and distinct roles for the sensing molecules in innate immunity. DNA viruses such as adenovirus stimulate the NLRP3-ASC-caspase-1 inflammasomes in vivo. The existence of a TLR-independent cytoplasmic DNA sensing molecule leading to type I IFN production was suggested from studies utilizing DNA viruses and bacteria. The mechanism of RNAi involves two steps. First, viral dsRNA is recognized by Dicer-like endonuclease family, which processes it into siRNA. Second, the siRNA are incorporated into RNA-induced silencing complex (RISC), which guide the RNase enzyme AGO to complementary sequences (viral RNA) for cleavage and degradation of viral RNA. Tetherin associates with lipid rafts and inhibits retrovirus particle release in the absence of Vpu. Vpu utilizes the beta-TrCP E3 ubiquitin ligase complex to induce endosomal trafficking events that remove tetherin from the cell surface, rendering it incapable of restricting the release of enveloped viruses.

This chapter predicts and highlights the genome functions of Tannerella forsythia related to bacterial community development. The major intent is to identify putative genes that are likely to be important in T. forsythia interactions with the members of the bacterial community. The chapter focuses on a predicted function derived from the recently completed T. forsythia genome. It was demonstrated that T. forsythia growth required an exogenous source of N-acetylmuramic acid (MurNAc). Investigation of the genome functions that assist T. forsythia in scavenging MurNAc from coinhabiting species could lead to the identification of novel mechanisms for MurNAc uptake in bacteria, as well as to the design of novel strategies for blocking MurNAc uptake by T. forsythia and controlling periodontitis. The chapter talks about surface components, surface layer (S-layer) glycoproteins, and LRR proteins. Only a few virulence factors have been identified in T. forsythia. The metabolic conditions of the host could influence T. forsythia growth and virulence gene expression. Recent studies have shown that expression of the T. forsythia virulence protein BspA and its homologues is affected in response to environmental cues. T. forsythia possesses several conjugative transposon (CTn) elements belonging to the Bacteroides CTnDOT family. In the near future, computational approaches supported by direct laboratory experimentation will be necessary to decipher some of the complex bacterial interactions.